1,3,5-Triazine-2,4-diamine, N2-[(1R,2S)-2,3-dihydro-2,6-dimethyl-1H-inden-1-yl]-6-(1-fluoroethyl)-

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

OECD 416, rat, 2-generation, oral: Parental: NOAELsystemic = 10.2 mg/kg bw/day; LOAELsystemic = 68.9 mg/kg bw/day; NOAELrepro ≥ 560.1 mg/kg bw/day; F1: NOAELrepro = 80.8 mg/kg bw/day; LOAELrepro = 355.8 mg/kg bw/day; F2: NOAELsystemic = 13.3 mg/kg bw/day; LOAELsystemic = 84.9 mg/kg bw/day

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Guidelines on Reproductive Toxicity Studies, 91/414/EEC

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: J.M.A.F.F. 12 Nousan No 8147

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Health Canada, Guidelines on Reproduction Toxicity Studies

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Wistar Hanover (Crl:WI(Glx/BRL/Han)IGS BR)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC
- Age at study initiation: (P) 8 wks; (F1) approx. 9 wks (initiation of second generation)
- Weight at study initiation: (P) Males: 203.6-261.1 g; Females: 136.2-176.0 g
- Fasting period before study: not applicable, feeding study
- Housing: Individually (except during the mating phase and as noted below for the F1 and F2 pups) in suspended stainless steel cages with deotized cage board in the bedding trays. During gestation and lactation, individual dams (and litters) were housed in polycarbonate cages with corn-cob bedding.
A single Nylabone® was placed in the cage of each animal (to provide environmental enrichment). Adult males were given the Nylabone® for the duration of the study except for the cohousing phase. Adult females were given the Nylabone® during the premating phase only. Pups passing vaginal patency and preputial separation were transferred to individual cages and provided a Nylabone®.
- Diet: ad libitum – Purina Mills Rodent Lab Chow 5002 meal
- Water: ad libitum – pressure-activated water nipples or water bottles
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%): 30-70
- Air changes (per hr): minimum of 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
The test compound was mixed directly with the feed. Treated diet was mixed at room temperature; aliquots of the chemical were taken from the original test batch and transferred to the mixing area. The control test diet was taken directly from the bag without mixing. A sample of each batch of feed mixed was taken and retained in the freezer until the study was complete and the analytical data were deemed satisfactory.
The concentration of the test substance in the feed, for the females only, was reduced during the lactation period (Days 0-21) by 50%. Samples from the first batch of adjusted feed for each dietary level were analyzed to measure the concentration. During the lactation phase, a substantial increase in food consumption is observed in all dams which results in greatly increased intake of test substance (normal occurrence). A decrease in the dietary concentration of the test substance offsets this increased food consumption, thereby maintaining an approximately constant test substance intake (mg/kg body weight/day) throughout the study.

Calculation of test substance intake:
Mean analytical concentration (ppm) specific for each phase / 1000 X mean weekly food consumption (g/kg bw/day) for each phase.

- Rate of preparation of diet (frequency): Replacement admixtures for each treatment group were prepared weekly (or at greater intervals, if within freezer stability limits) during the entire study. Additionally, the entire batch of replacement admixture for each treatment group was used for subsequent weekly feeding if within freezer stability limits.
- Mixing appropriate amounts with (Type of food): Purina Mills Rodent Lab Chow 5002 meal.
- Storage temperature of food: Stored at freezer conditions until presented to the animals.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 consecutive days
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): Females found to be inseminated were placed in a polycarbonate nesting cage.
- Any other deviations from standard protocol: In order to evaluate those females which might have been inseminated without exhibiting sperm in the vaginal smear or an internal vaginal plug, all remaining females were placed in polycarbonate nesting cages, following the 14-day mating period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of test substance in the various test diets were analytically verified for batches intended for weeks 1, 2, 3 and at monthly intervals thereafter (Bayer Crop science LP, Residue and Environmental Chemistry Group, 17745 S. Metcalf, Stilwell, KS). The homogeneity and stability of the test substance when mixed in the rodent feed were characterized, as well.
Homogeneity:
The mean concentrations of test substance in the feed, sampled from three distinct layers in the mixing bowl and containing a nominal concentration of either 20- or 10000-ppm, were determined to be 18.0 ppm (range 16.9-19.1 ppm; %RSD = 3.2) and 9079 ppm (range 8663-9443 ppm; %RSD = 2.9), respectively. Based on a %RSD <10%, test substance was judged to be homogeneously distributed in the feed over a concentration range of 20-10000 ppm (Jensen, 2007).
Stability:
Following 7 days of room temperature storage, the analytically determined concentration of the AI of the test substance in the 20- and 10000-ppm admixture was determined to be 19.0 ppm (18.0 ppm on Day 0) and 9376 ppm (8991 ppm on Day 0), respectively. Following 63 days of freezer storage, the analytically determined concentration of the AI of the test substance in the 20- and 10000-ppm admixtures was determined to be 18.4 ppm (18.0 on Day 0) and 9250 ppm (9079 on Day 0), respectively. The test substance mixed in rodent ration was judged to be stable at room temperature for at least seven days and following freezer storage for a minimum of 63 days, over a concentration range of 20-10000 ppm (Jensen, 2007).
Concentration:
Mean analytical concentrations for each dose group were 154, 1045, 4143 (high dose reduced from 8000 to 4000 ppm due to marked toxicity) and 8143 ppm, ranging from 102–105% of the corresponding nominal concentrations of 150, 1000, 4000 and 8000 ppm, respectively. During lactation the concentration of the test substance in the feed for the females was adjusted by 50%. During the lactation phase, a substantial increase in food consumption is observed in all dams which results in greatly increased intake of test substance (normal occurrence). A decrease in the dietary concentration of the test substance offsets this increased food consumption, thereby maintaining an approximately constant test substance intake (mg/kg bw/day) throughout the study. Mean analytical concentrations for each dose group during lactation were 79.3, 520, 2059 and 4143 ppm, ranging from 103-106% of the corresponding nominal concentrations of 75, 500, 2000 and 4000 ppm, respectively. The AI of the test substance was not detected in the control diet. Mean recovery was 102% and ranged from 95-112% for rodent ration spiked with 75, 150, 500, 1000, 2000, 4000 or 8000 ppm of test substance (Moore and Neal, 2008).

Duration of treatment / exposure:

approx. 39 weeks (10 weeks prior to mating, 14 days mating, approximately 22 days gestation, 21 days lactation until weaning; F1-pups 6 weeks after weaning prior to initiation of second generation. F2-pups were maintained after weaning for evaluation of developmental landmarks until PND 70.)

Frequency of treatment:

continuously

Details on study schedule:

- F1 parental animals not mated until 6 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 18 weeks (P); 9 weeks (F1)

Remarks:

Doses / Concentrations:
150, 1000, 8000 ppm (corresponding to 10.2, 68.9, 560.1 mg/kg bw/day for males and 12.6, 83.2, 656.2 mg/kg bw/day for females during premating and 11.9, 80.3, 637.4 mg/kg bw/day for females during gestation)
Basis:
nominal in diet
P-generation premating/gestation

Remarks:

Doses / Concentrations:
75, 500, 4000 ppm (corresponding to 13.7, 87.7, 730.5 mg/kg bw/day)
Basis:
nominal in diet
P-generation lactation (reduction of dietary concentration due to increased food comsumption during lactation)

Remarks:

Doses / Concentrations:
150, 1000, 4000 ppm (corresponding to 10.6, 69.6, 317.6 mg/kg bw/day for males and 13.1, 87.2, 355.5 mg/kg bw/day for females during premating and 11.1, 80.8, 355.8 mg/kg bw/day for females during gestation)
Basis:
nominal in diet
F1-generation premating/gestation

Remarks:

Doses / Concentrations:
75, 500, 2000 ppm (corresponding to 13.3, 84.9, 363.1 mg/kg bw/day)
Basis:
nominal in diet
F1-generation lactation (reduction of dietary concentration due to increased food consumption during lactation)

No. of animals per sex per dose:

30

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
Based upon the preliminary results which emerged in the rat over the course of a pilot reproductive toxicity testing study conducted with the test chemical at doses of 0, 200, 1,000, 3,000 and 8,000 ppm (Milius, 2008). In that study, clinical observations considered to be attributed to the test article were not observed at any dietary level tested. Declines in body weight for the females of the 8000 ppm dose group were observed throughout the premating, gestation and lactation phases. Females of the 8000 ppm dose group also exhibited declines in food consumption during the premating period with initial declines observed during the first weeks and continuing through week eight. No test article-related effect on body weight or food consumption was observed in the males at any dietary level tested. In the 8000 ppm dose group, declines in female terminal body weights (-7%) and uterine weights (absolute, -41%; relative, -37%) were observed, as well as a trend toward increased thyroid weights (absolute, +29%; relative, +44%). Adult male organ weight effects included increased absolute and relative liver weights in both the 3000 and 8000 ppm dose groups and increased absolute and relative adrenal and kidney weights in the 8000 ppm dose group. Males showed no effect on terminal body weight at any dietary level tested. In the 8000 ppm dose group, female pup weight declines were observed on Day 21 (7%) with a decline in gain observed Days 7-14 (8%) and Days 14-21 (14%). Organ weight declines were observed in the spleen (absolute, -21%; relative, -15%) and uterus (absolute, -19%; relative, -13%) of the female pups of this same dose group. Body and/or organ weight effects were not observed in the male pups at any dietary level tested. Based on these interim results, the doses selected for the two-generation reproduction toxicity study are 0, 150, 1000 and 8000 ppm. This dose range is intended to produce evidence of toxicity at the highest dietary concentration and no parental or reproductive effects at the lowest dietary concentration.

Positive control:

none

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (AM and PM) and once daily on weekends and holidays.
- Cage side observations included: Mortality, morbidity, behavioral changes, signs of difficult or prolonged delivery, and overt toxicity by viewing the animal in the cage. In the event a possible clinical sign was observed during the cageside evaluation, the animal was removed from the cage and a detailed assessment conducted.

DETAILED CLINICAL OBSERVATIONS: Yes, including observation of the animal in the cage and removal of the animal to perform a physical examination.
- Time schedule: at least one per week throughout the entire in-life phase of the study.

BODY WEIGHT: Yes
- Time schedule for examinations: Once per week during the 10-week premating period, during mating and until sacrifice for males and unmated females. During gestation, dam body weight was measured on days 0, 6, 13 and 20; during lactation, dam body weight was measured on days 0, 4, 7, 14 and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, once per week during the 10-week premating period, during gestation and la. During mating, food consumption was not measured. During lactation exept week one of lactation
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as mean daily intake for each phase from the consumption and body weight data: Yes, calculation for test substance intake is: Mean analytical concentration (ppm) specific for each phase / 1000 X mean weekly food consumption (g/kg/body weight/day) for each phase.

Oestrous cyclicity (parental animals):

The estrous cycle (determined by examining daily vaginal smears) was characterized for all P- and F1-generation females, over a three-week period prior to mating. Additionally, the estrous cycle stage was determined for all females just prior to termination.

Sperm parameters (parental animals):

Parameters examined in P and F1 male parental generations (one testis and one epididymis per male):
High dose + control groups: Sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm morphology
All groups: Sperm motility

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); grossly abnormal pups underwent a gross internal and external examination, and all culled pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
Number of live pups (PND 0-21), pup weight, sex of pups, stillbirths, postnatal mortality (PND 0-21), presence of gross anomalies, gross abnormalities, preputial separation, vaginal patency

GROSS EXAMINATION OF DEAD PUPS:
Yes, grossly abnormal pups for external and internal abnormalities; possible cause of death was determined for pups born or found dead or terminated in moribund condition.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals as soon as possible after the last litters in each generation were produced.
- Maternal animals: All surviving animals after the last litter of each generation was weaned (lactation day 22).

GROSS NECROPSY
- Gross necropsy of males consisted of: Determination of terminal body weights and recording of all gross pathologic alterations, weighing designated organs, and saving all gross lesions.
-Gross necropsy of females consisted of: Gross external examination of each dam (both P- and F1-generations). Terminal body weights were measured and the recording of all gross pathologic alterations, weighing designated organs, and saving all gross lesions was conducted on all females. The uterus was excised and the implantation sites, if present, were counted. Females that were sperm positive and/or had an internal vaginal plug but did not deliver were sacrificed after gestation day 24. Females that were never observed as being inseminated and/or with an internal vaginal plug and did not deliver at least 24 days after the completion of the mating phase, were sacrificed and necropsied. A gross necropsy was performed on these animals as described above. In addition, patency of the cervical/uterine os (opening) in these females was examined via flushing of the uterine horns with 10% buffered formalin.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated were prepared for microscopic examination and weighed, respectively:
Organ weights: Brain, pituitary, liver, kidney, spleen, thyroid, thymus, adrenal, cervix, epididymis, coagulating gland, ovary, prostate, seminal vesicle, testis, uterus, vagina, epididymis cauda.
Micropathology: Pituitary, liver, kidney, spleen, adrenal, cervix, epididymis, coagulating gland, oviduct, prostate, seminal vesicle, testis, uterus, vagina, epididymis cauda, gross lesions.
Additionally, the lung and the physical identifier were collected, but not subjected to any further determination.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: Macroscopical examination and determination of organ weight. One pup/sex/litter for each generation had tissues collected and evaluated for any structural abnormalities or pathological changes. Organs collected: Brain, thymus, gross lesions, uterus, ovary, vagina, cervix, oviduct, testis, epididymis, prostate, coagulating gland, seminal vesicle.

GROSS NECROPSY
Pups found dead or terminated in moribund condition underwent a gross necropsy for possible defects and/or cause of death.
- Gross necropsy consisted of external and internal examinations.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated were prepared for microscopic examination and weighed, respectively:
Micropathology: Gross lesions, uterus, ovary, vagina, cervix, oviduct, testis, epididymis, prostate, coagulating gland, seminal vesicle.
Organ weight: Brain, spleen, thymus, uterus.

Statistics:

The data were analyzed using applications provided by DATATOX (Instem Computer Systems), SAS (SAS Institute, Inc.), or TASC (Toxicology Analysis Systems Customized, 1993). Parametric data (including body weight and food consumption, as well as body weight gain for pups and females during gestation were analysed using a univariate Analysis of Variance (ANOVA), and if significant differences were observed, a Dunnett’s Test was performed. Nonparametric data (e.g., number of estrous cycles, litter size, and number of implantation sites) were first analysed by the Kruskal-Wallis test and then subjected to Dunn’s Test if significant differences were identified. Nonparametric dichotomous data (e.g. fertility and gestation indices) were initially analysed by the Chi-Square Test, and if significance was observed between groups, then by the Fisher’s Exact Test with the Bonferroni adjustment. To the extent possible, the frequency of gross lesions was first examined visually, then, in the event of questionable distribution, by statistical analysis using the Chi-Square and Fisher’s Exact tests. Differences between the control and test substance-treated groups were considered statistically significant when p≤0.05 or p≤0.01.

Reproductive indices:

Mating index, fertility index, gestation index,

Offspring viability indices:

Birth index, livebirth index, viability index, lactation index, gestation length

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

adverse

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

adverse

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

adverse

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

adverse

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

secondary effect in F1 parents due to severe toxicity

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
P-generation: There were no mortalities at any dietary level tested. A test substance-related increase in the incidence of coarse tremors was observed in the P-generation females of the 8000 ppm dose group. There were no other clinical observations considered to be test substance-related in the females at any other dietary level or in the males at any dietary level tested.

F1-pups (PND 0-21): There was no test substance-related effect on the viability of the pups prior to weaning at any dietary level tested. Based on clinical observations observed in the F1-pups after day 21, the clinical observations of urine stains and nasal stains in the 8000 ppm dose group can not be ruled out as test substance-related. There were no test substance-related clinical observations observed at any other dietary level tested.

F1-pups (PND 22- 61): Prior to reducing the dose from 8000 ppm to 4000 ppm, F1-pups were severely affected by the test substance at this high dietary level. Two pups from one litter were sacrificed in a moribund condition on their PND 23, and two pups from another litter were found dead on their PND 27. Many pups from the majority of the litters exhibited severe clinical signs. These observations included increased activity, increased reactivity, myo-clonus (jerking movements), tremors, distended abdomen, labored breathing, diarrhea, soft stool, and various stains (perianal, urine, and nasal). There were no test substance-related effects observed on clinical observations at any other dietary level tested.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Males:
P-generation males of the 8000 ppm dose group exhibited a slight decline in body weight gain (declined 6.6%) after 14 weeks of exposure when compared to controls. There were no test substance-related effects on absolute body weight at any dietary level tested. Incidental increases in absolute body weight and body weight gain occurred in the 150 and 1000 ppm dose group. This finding did not show a dose response relationship and is more probably due to animals being a little heavier (although within ±20% of the mean for all males at randomization) at start of treatment.

A statistically significant increase in food consumption was observed in the males of the 8000 ppm dose group on a g/kg bw/day basis beginning week 6 of premating (mean increase of 6.6%). Food consumption in this dose group was comparable to the controls on a g/animal/day basis. Incidental declines in food consumption were observed for the males of the 1000 and 8000 ppm dose groups during the first week of premating and are considered to be due to initial palatability of the test substance. By the second week of premating the food consumption values for males were comparable to controls.

F1-generation males of the 4000 ppm dose group showed a decline in absolute body weight beginning on day 0 (declined 21.2% compared to controls) and continuing throughout the 14-week period, ending in a decline at that time of 10.1%, relative to controls. This decline in absolute body weight is considered to be due to the significant declines in weight of these animals when they were pups. In this same dose group, the absolute body weight gain was increased from controls 12.3%. Also noted was a significant increase in food consumption (mean increase of 17.0%), relative to controls. These findings exhibit some compensation in growth after lowering the dose to 4000 ppm. Body weight effects were not observed at any other dietary level tested. There were no effects observed on food consumption at any dietary level tested that were considered to be attributed to the test substance.

Females (Premating):
P-generation females of the 8 000 ppm dose group exhibited statistically significant declines in absolute body weight beginning the second week of treatment and continuing throughout the 10-week premating period (mean decline weeks 2-10 was 7.8%). Body weight gain was declined 27.4% in this same dose group when compared to controls. Slight declines in body weight were observed in the 1000 ppm dose group beginning week 5 of treatment with statistical significance noted only during week 8. Body weight effects were not observed at the 150 ppm dietary level.

Beginning week 4 of treatment, sporadic declines in food consumption were observed in both the 1000 and 8000 ppm dose groups on a g/kg bw/day basis. Declines in food consumption on a g/animal/day basis were also noted in these same dose groups with more consistent declines in the 8000 ppm dose group (mean decline weeks 4-10 of 10.1%). No test substance-related findings were observed on food consumption at the 150 ppm dietary level. Incidental declines in food consumption were observed for the females of the 8000 ppm dose group during the first week of premating and are considered to be due to initial palatability of the compound. Food consumption was comparable to controls by the second week prior to the test substance-related declines beginning week 4.

F1-generation females of the 4000 ppm dose group showed a decline in absolute body weight beginning on day 0 (declined 13.2% compared to controls) and continuing throughout the premating period ending in a decline at that time of 10.7%, relative to controls. This decline in absolute body weight is considered to be partially due to the significant declines in weight of these animals when they were pups. In this same dose group, the absolute body weight gain was comparable to controls.

Food consumption evaluation for the females of this level showed sporadic increases on a g/kg bw/day basis but overall the food consumption on a g/animal/day basis was decreased when compared to controls. Based on these findings, the females seem to be having a more difficult time compensating after lowering the dose to 4000 ppm. Body weight effects were not observed at any other dietary level tested. There were no effects observed on food consumption at any other dietary level tested.

Females (Gestation):
P-generation females of the 8000 ppm dose group showed statistically significant body weight declines during gestation beginning day 0 (decline of 8.8%) and continuing throughout gestation. By Day 20 of gestation decline in body weight, relative to controls, was 14.8%. Body weight gain throughout gestation was declined 29.7%. Body weight effects were not observed at any other dietary level tested.

There were no effects on food consumption observed at any dietary level tested.

F1-generation females of the 4000 ppm dose group showed statistically significant body weight declines during gestation beginning day 0 (decline of 10.2%) and continuing throughout gestation. By day 20 of gestation decline in body weight, relative to controls, was 12.7%. Body weight gain throughout gestation was declined 19.1%. In the 1000 ppm dose group, nonstatistical declines in body weight were observed beginning on day 0 with statistical declines by day 20. There was no effect on body weight gain in the 1000 ppm dose group. Body weight effects were not observed at the 150 ppm dietary level.

Food consumption on a g/kg bw/day basis was increased in the 4000 ppm dose group with significance observed on days 13-20 (increased 12%, relative to controls). There were no effects on food consumption considered to be test substance-related at any other dietary level tested.

Females (lactation):
P-generation females of the 8000 ppm dose group demonstrated statistically significant body weight declines, relative to control, throughout lactation (mean decline days 0-21 of 14.3%). Body weight effects were not observed at any other dietary level tested.

In the 8000 ppm dose level food consumption on a g/animal/day basis was statistically declined days 4-21 (mean decline of 14.7%), relative to the controls. There were no test substance-related effects on food consumption observed at any other dietary level tested.

F1-generation females of the 4000 ppm dose group showed statistically significant body weight declines, relative to control, throughout the lactation period (days 0-21; mean decline of 10.5%). There were no test substance-related effects on body weight at any other dietary level tested, although trends of lower body weight when compared to the controls were noted in the 1000 ppm dose group.

In the 4000 and 1000 ppm dose levels, food consumption on a g/animal/day basis was statistically declined, relative to controls, on days 4-21 (mean decline of 8.9 and 13%, respectively). There were no food consumption effects considered to be test substance-related at the 150 ppm dietary level.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Based on food consumption, body weight and dietary analyses results, the doses expressed as mean daily mg test substance/kg body weight during the pre-mating period (10 weeks for males and females) were calculated according to the following formula: Mean analytical concentration (ppm) specific for premating / 1000 X mean weekly food consumption (g/kg/body weight/day) during premating. Results can be found above under "Doses/concentrations".

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Results from the evaluation of vaginal smears in both the P- and F1-generation females did not indicate any test substance-related findings at any dietary level tested.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
There were no test substance-related effects observed on any sperm parameter evaluated at any dietary level tested for either generation. An isolated decline in testicular counts was observed in the P-generation males of the 8000 ppm dose group and is considered to be incidental to treatment based on the following. There was no concomitant reduction in epididymal counts, in fact the epididymal count for this generation was higher in the 8000 ppm dose group, relative to control. Also, there was no effect on testicular weight, no associated micropathology in the testes, nor was there any reproductive consequence. A decline in testicular count was not observed at the 4000 ppm dose level of the F1-generation.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
P-generation:
Overall reproductive performance was not affected for any parameter (e.g., mating, fertility or gestation indices, days to insemination, gestation length, or the median number of implants) at any dietary level tested.

F1-generation:
A decrease in the mean number of implantation sites was noted in the 4000 ppm dose group. This finding corresponds to a slight decrease in litter size (mean of 9.3, relative to controls) observed in this same dose group. The mean litter size is just outside of historical control values for this laboratory in this strain of rat (range 9.4-12.8). These reproductive findings are considered to be due to the significant toxicity observed in these animals during the sensitive period of growth and development when they were pups as discussed under F1-Offspring. It is also taken into consideration that there were no reproductive effects in the P-generation adults when exposed to the higher concentration of 8000 ppm. Overall reproductive performance was not affected for any other parameter (e.g., mating, fertility or gestation indices or days to insemination) at any other dietary level tested.

ORGAN WEIGHTS (PARENTAL ANIMALS)
P-Generation Adults:
In the 8000 ppm dose group, females exhibited significant declines in terminal body weight (mean decline of 11.46%, relative to controls). Terminal body weight effects were not observed at any other dietary level tested in the females. There were no test substance-related effects on terminal body weights in the P-generation males at any dietary level tested. Organ weight changes considered to be test substance-related in the 8000 ppm dose group included increased absolute and relative liver and kidney weights in the males and decreased absolute and relative spleen and uterus weights in the females. In the 1000 ppm dose group, increased absolute and relative liver weights were observed in the males. No other organ weight changes considered to be test substance-related were observed.

F1-Generation Adults:
Statistically significant terminal body weight declines, relative to controls, were observed in the 4 000 ppm dose group of both genders (males decreased 10.6% and females decreased 8.5%). Terminal body weight effects were not observed at any other dietary level tested. There were no organ weight changes considered to be directly test substance-related in either the males or females of the F1-generation. The decrease noted in the 4000 ppm female pituitary weights (both absolute and relative) was not associated with morphological changes and is of equivocal biological significance. This finding may also be associated with the severe toxicity these animals suffered from during the sensitive growth and development of the brain when they were pups.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no test substance-related gross necropsy findings observed at any dietary level tested in either generation.

HISTOPATHOLOGY (PARENTAL ANIMALS)
P-Generation:
Minimal to moderate centrilobular hepatocellular hypertrophy extending into the midzonal lobular region in some animals was noted in the livers of 1000 and 8000 ppm males and 8000 ppm females. In the 8000 ppm male kidneys, minimal to slight “hyaline degeneration” was noted in the renal cortices, principally within the proximal convoluted tubules of the kidney. In addition, there was an increase in the background incidence of minimal to slight “tubular regeneration” (focal or multifocal) within the renal cortex. In the females of the 8000 ppm dose group there was a subtle, generally minimal to mild increase in “cytoplasmic vacuolation” of the zona glomerulosa (outer zone) of the adrenal cortices. All other microscopic lesions were considered to be incidental and/or background and were not considered to be test substance-related.

F1-generation:
Minimal to moderate centrilobular hepatocellular hypertrophy extending into the midzonal lobular region in some animals was noted in the livers of 4000 ppm males and females. In the 4000 ppm male kidneys, minimal to slight “hyaline degeneration” was noted as described for the males of the P-generation. All other microscopic lesions were considered to be incidental and/or background and were not considered to be test substance-related.

OTHER FINDINGS (PARENTAL ANIMALS)
Ovarian Follicle Counts from F1-Generation Females:
None of the mean primordial (preantral), antral, or corpora lutea follicular counts were statistically different from controls. The nonstatistical decrease in the mean numbers of corpora lutea of the 4000 ppm dose group were not associated with abnormal morphologic changes. All stages of corporal luteal development and regression were present. This finding could be associated with the slightly smaller litter size observed in the F1-females and may have been caused by the severe toxicity to these females as pups when exposed to the 8000 ppm dietary level.

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

150 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse systemic effects observed

Remarks on result:

other: corresponding to 10.2 and 12.6 mg/kg bw/day for males and females, respectively

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Remarks on result:

other: corresponding to 68.9 and 83.2 mg/kg bw/day for males and females, respectively

Dose descriptor:

NOAEL

Remarks:

reproductive

Effect level:

>= 8 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects on reproduction observed

Remarks on result:

other: corresponding to 560.1 and 637.4 mg/kg bw/day for males and females, respectively

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

adverse

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

adverse

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

adverse

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

secondary effects

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

secondary effects

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

VIABILITY (OFFSPRING)
F1-pups (PND 0-21): There was no test substance-related effect on the viability of the pups prior to weaning at any dietary level tested.

F1-pups (PND 22- 61): Prior to reducing the dose from 8000 ppm to 4000 ppm, F1-pups were severely affected by the test substance at this high dietary level. Two pups from one litter were sacrificed in a moribund condition on their PND 23, and two pups from another litter were found dead on their PND 27. Many of the remaining pups from the majority of the litters exhibited severe clinical signs, as described below. There were no test substance-related effects observed on clinical observations at any other dietary level tested.

F2-pups (PND 0-70): There were no test substance-related effects on the viability of the pups at any dietary level tested.

CLINICAL SIGNS (OFFSPRING)
F1-pups (PND 0-21): Based on clinical observations observed in the F1-pups after day 21, the clinical observations of urine stains and nasal stains in the 8000 ppm dose group can not be ruled out as test substance-related. There were no test substance-related clinical observations observed at any other dietary level tested.

F1-pups (PND 22- 61): Prior to reducing the dose from 8000 ppm to 4000 ppm, F1-pups were severely affected by the test substance at this high dietary level, as reflected by the observed mortality. Many pups from the majority of the litters exhibited severe clinical signs. These observations included increased activity, increased reactivity, myo-clonus (jerking movements), tremors, distended abdomen, labored breathing, diarrhea, soft stool, and various stains (perianal, urine, and nasal). There were no test substance-related clinical signs observed at any other dietary level tested.

F2-pups (PND 0-70): There were no test substance-related clinical observations observed at any dietary level tested. The outward rotation of the forepaws noted in 4 pups from 1 litter of the 4000 ppm dose group is considered to have been due to the extreme toxicity presented in the parents of this litter when they were pups and not a direct result of the test substance. In addition, this finding was not noted in the F1-pups when exposed to the higher dose of 8000 ppm. Misshapen forelimb and/or deformed forelimbs noted in 1 pup from 1 control litter and 3 pups from 3 litters of the 1000 ppm dose group was due to damage during the tattooing process.


BODY WEIGHT (OFFSPRING)
Lactation until weaning:
F1-Pups: Pup body weights at birth for all three treated groups were comparable to the control group. In the 8000 ppm dose group, statistically significant declines in body weight were observed by Day 4 and continued throughout lactation (mean decline days 4-21 of 17.1%). Overall body weight gain was declined 22.6%, relative to control. Declines in pup body weight were not observed at any other dietary level tested.

F2-Pups: Pup body weights at birth for all three treated groups were comparable to the control group. In the 4000 ppm dose group, declines in body weight were observed by day 21 (mean decline of 5.9% in males and 5.8% in females). Overall body weight gain was declined 6.7%, relative to control. In the 1000 ppm dose group, no effect on absolute body weight was observed. Changes in body weight gain, relative to the controls, were observed on lactation days 14-21 in both the 1000 (more significantly with the females) and 4000 ppm dose groups. No declines in pup body weight were observed at the 150 ppm dietary level.

Juvenile pups (after weaning PND 21):
F1-juveniles of the 8000 ppm dose group (dose was decreased to 4000 ppm between the pup age range of 26-38 days old) exhibited significant declines in body weight, relative to controls, upon passing criteria for balanopreputial separation and vaginal patency. Male body weights were declined 7.3% and female body weights were declined 13.6%. There was no effect on body weight at passing these developmental landmarks, compared to the control group, at any other dietary level tested.

F2-Juveniles: There was no effect on body weight, relative to controls, upon passing criteria for balanopreputial separation and vaginal patency for the F2-juveniles at any dietary level tested.

SEXUAL MATURATION (OFFSPRING)
F1 pups: Significant delays in balanopreputial separation and vaginal patency were observed in the 8000 ppm dose group. The average male passed criteria for balanopreputial separation 9.3 days later than control males with the average female passing criteria for vaginal patency 6.8 days later than control females. Delays in passing these developmental criteria are considered to be test substance-related but these delays are considered to be exacerbated by the severe toxicity observed in these pups, exhibited by the clinical observations and significant body weight declines during the lactation period prior to decreasing the dose to 4000 ppm. Delays in balanopreputial separation and vaginal patency were not observed at any other dietary level tested.

F2-pups - Significant delays in balanopreputial separation and vaginal patency were observed in the 4000 ppm dose group. The average male passed criteria for balanopreputial separation 3.4 days later than control males with the average female passing criteria for vaginal patency 2.4 days later than control females. Delays in passing these developmental criteria are considered to be test substance-related but also correlate with pup body weight declines during lactation. Delays in balanopreputial separation and vaginal patency were not observed at any other dietary level tested.

ORGAN WEIGHTS (OFFSPRING)
F1-pups: Organ weight changes were observed on brain, spleen and thymus in both genders of the 8000 ppm dose group. These organ weight effects are considered to be secondary to the statistically significant pup weight declines observed during lactation at this dose level. Organ weight effects were not evident at any other dietary level tested.

F2-pups: Organ weight changes were observed on the brain and spleen in both genders of the 4000 ppm dose group. Spleen weights were also decreased in the female pups of the 1000 ppm dose group. These organ weight effects are considered to be secondary to the statistically-significant pup weight declines observed during lactation at this dose level. Organ weight effects were not evident at the 150 ppm dietary level.

GROSS PATHOLOGY (OFFSPRING)
There were no test substance-related gross necropsy findings observed at any dietary level tested in either the F1- or F2-pups.

HISTOPATHOLOGY (OFFSPRING)
There were no test substance-related microscopic findings observed at any dietary level tested in either the F1- or F2-pups.

Dose descriptor:

NOAEL

Remarks:

systemic

Generation:

F1

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse systemic effects observed

Remarks on result:

other: corresponding to 69.6 and 80.8 mg/kg bw/day for males and females, respectively

Dose descriptor:

LOAEL

Remarks:

systemic

Generation:

F1

Effect level:

4 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: corresponding to 317.6 and 355.8 mg/kg bw/day for males and females, respectively

Dose descriptor:

NOAEL

Remarks:

reproductive

Generation:

F1

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects on reproduction observed

Remarks on result:

other: corresponding to 80.8 mg/kg bw/day

Dose descriptor:

LOAEL

Remarks:

reproductive

Generation:

F1

Effect level:

4 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: number of implantation sites; litter size; other: number of corpora lutea; secondary effects due to severe toxicity

Remarks on result:

other: corresponding to 355.8 mg/kg bw/day

Dose descriptor:

NOAEL

Remarks:

reproductive

Generation:

F1

Effect level:

8 000 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effects on reproduction observed

Dose descriptor:

NOAEL

Remarks:

systemic

Generation:

F2

Effect level:

150 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse systemic effects observed

Remarks on result:

other: corresponding to 13.3 mg/kg bw/day

Dose descriptor:

LOAEL

Remarks:

systemic

Generation:

F2

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Remarks on result:

other: corresponding to 84.9 mg/kg bw/day

Reproductive effects observed:

yes

Lowest effective dose / conc.:

4 000 ppm

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

not specified

Relevant for humans:

not specified

CONCLUSION

For males, the parental systemic LOAEL is 1000 ppm (68.9 mg/kg bw/day), based on increased liver weights associated with centrilobular hepatocellular hypertrophy in the P-generation, and the systemic NOAEL is 150 ppm (10.2 mg/kg bw/day).

For females, the parental systemic LOAEL is 1000 ppm (83.2 mg/kg bw/day), based on decreased body weight and food consumption in the P-generation, and the systemic NOAEL is 150 ppm (12.6 mg/kg bw/day).

For males and females of the P-generation, the reproductive NOAEL is greater than 8000 ppm (≥560.1 mg/kg bw/day and ≥637.4 mg/kg bw/day, respectively), based on no adverse reproductive findings observed. For F1-males the reproductive NOAEL is greater than 8000 ppm, as well, as no adverse effects were observed, either.

For females of the F1-generation the reproductive LOAEL is 4000 ppm (355.8 mg/kg bw/day), based on decreased number of implants, corpora lutea and smaller litter size, considered to be secondary to severe toxicity in these females as pups. The reproductive NOAEL is 1000 ppm in F1-females (80.8 mg/kg bw/day).

The systemic LOAEL for the F2-generation is 1000 ppm (84.9 mg/kg bw/day), according to the study authors. The systemic LOAEL is based on decreased body weight gain with secondarily mediated spleen weight declines in F2-female pups. The systemic NOAEL for the F2-generation is 150 ppm (13.3 mg/kg bw/day).

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

80.8 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.7, of Regulation (EC) No 1907/2006.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

There is a Two-Generation Reproduction Toxicity Study available which was conducted in rats according to OECD guideline 416 under GLP conditions.

In this study the parental animals received doses of 150, 1000 and 8000 ppm during the premating period and gestation (corresponding to 10.2, 68.9 and 560.1 mg/kg bw/day for males and 12.6, 83.2 and 656.2 mg/kg bw/day for females during premating, and 11.9, 80.3 and 637.4 mg/kg bw/day for females during gestation). During the lactation period, the dietary concentrations of the females were reduced to 75, 500 and 4000 ppm (corresponding to 13.7, 87.7 and 730.5 mg/kg bw/day) to compensate for the increased food consumption during lactation and prevent an increase in compound intake. Due to marked clinical signs in the high-dose animals of the F1-generation the dose level was reduced from 8000 to 4000 ppm after weaning in the age range of 26 to 38 days; thus, during the premating phase and during gestation the parental animals of the F1 generation were exposed to dietary concentrations of 150, 1000 and 4000 ppm (corresponding to 10.6, 69.9 and 317.6 mg/kg bw/day for males and 13.1, 87.2 and 355.5 mg/kg bw/day for females during premating, and 11.1, 80.8 and 355.8 mg/kg bw/day for females during gestation). During the lactation period, the dietary concentrations of the F1 parental females were reduced, as well, to 75, 500 and 2000 ppm (corresponding to 13.3, 84.9 and 363.1 mg/kg bw/day) to compensate for the increased food consumption during lactation.

The parental males exhibited increased liver weights, associated with centrilobular hepatocellular hypertrophy, which were considered adverse by the authors, at a dose level of 1000 ppm. Therefore, the parental systemic LOAEL for males is determined as 1000 ppm (68.9 mg/kg bw/day); the corresponding systemic NOAEL is 150 ppm (10.2 mg/kg bw/day). For females the parental systemic LOAEL is a dietary level of 1000 ppm (83.2 mg/kg bw/day), as well, based on decreased body weight and food consumption (according to the authors of the study); the corresponding systemic NOAEL for parental females is also 150 ppm (12.6 mg/kg bw/day).

Both males and females of the F1-generation demonstrate severe signs of toxicity at a dietary concentration of 4000 ppm (m: 317.6 mg/kg bw/day; f: 355.8 mg/kg bw/day), comprising clinical signs, increased mortality and moribundity, effects on body weight and food consumption. No adverse effects were observed at any other dietary level in the F1-generation; the systemic LOAEL for the F1-generation is a dietary level of 4000 ppm, the corresponding systemic NOAEL for the F1-animals is 1000 ppm (m: 69.6 mg/kg bw/day; f: 80.8 mg/kg bw/day).

The systemic LOAEL for the F2-generation is 1000 ppm (84.9 mg/kg bw/day), according to the study authors. The systemic LOAEL is based on decreased body weight gain with secondarily mediated spleen weight declines in F2-female pups. The corresponding systemic NOAEL for the F2 generation is 150 ppm (13.3 mg/kg bw/day).

The reproductive NOAEL for the males of the P- and the F1-generation is greater than 8000 ppm (≥560.1 mg/kg bw/day); no adverse effects on reproductive parameters were observed in any generation at any dose levels tested in males. For the females of the P-generation the reproductive NOAEL is also greater than 8000 ppm (≥637.4 mg/kg bw/day for females) in the diet, no adverse effects on reproductive parameters were observed in this generation at any dietary level tested. In contrast, for females of the F1-generation the reproductive LOAEL is 4000 ppm (355.8 mg/kg bw/day) based on decreased number of implants, corpora lutea and smaller litter size. However, as these reproductive effects were observed in cunjunction with the severe signs of toxicity described above, these effects are considered to be secondary to systemic toxicity. The reproductive NOAEL for the F1-females is 1000 ppm (80.8 mg/kg bw/day).

Effects on developmental toxicity

Description of key information

OECD 414, rat, prenatal, GD6-20, oral: NOAELmaternal ≥ 200 mg/kg bw/day; NOAELdevelopmental ≥ 200 mg/kg bw/day 
OECD 414, rabbit, prenatal, GD6-28, oral: NO(A)ELmaternal = 25 mg/kg bw/day; LOAELmaternal = 60 mg/kg bw/day; NO(A)ELdevelopmental ≥ 60 mg/kg bw/day

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: M.A.F.F. in Japan notification 12 Nousan No 8147, (November, 2000)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River laboratories, St Germain-sur-l'Arbresle, France.
- Age at study initiation: adult virgin, not further specified
- Weight at study initiation: 239-299 g (females selected for study)
- Fasting period before study: not applicable
- Housing: Pregnant females were housed individually in suspended, stainless steel, wire mesh cages.
- Diet (e.g. ad libitum): Certified rodent pelleted and irradiated diet A04C-10 from S.A.F.E. (Scientific Animal Food and Engineering, Augy, France) was distributed ad libitum. Feed was stored in an identified room, controlled for temperature and humidity. Diet was used only until the date of expiry.
- Water (e.g. ad libitum): Water from the municipal supply was provided ad libitum with an automatic watering system.
- Acclimation period: at least 12 days prior to mating

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 0.5% methylcellulose 400

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The appropriate amount of test substance was suspended (w/v) in an aqueous solution of methylcellulose 400 (Fluka, Mulhouse, France) at 0.5% and stored at approximately 5 °C (± 3 °C). Formulations were prepared twice (F1 and F2) during the study. The suspensions were mixed continuously before and during treatment with an electromagnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.5% aqueous methylcellulose 400; methylcellulose is commonly used as thickener or emulsifier in various food and cosmetic applications and toxicological studies to generate appropriate consistency.
- Concentration in vehicle: 0, 1.0, 2.5 and 20.0 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity of the suspensions was checked on the first formulation (F1) for the lowest and the highest concentrations (1 and 20 mg/mL). The mean values obtained from the homogeneity check were used as measured concentrations. In addition, the intermediate concentration of the first formulation (F1) and all concentrations of the second formulation (F2) were checked. Stability of the test substance in suspension in the vehicle at concentrations of 0.127 and 250 g/L was determined in a previous study (SA 04129) and was found to be stable for 30 days under similar conditions to those of the current study.

Homogeneity and concentration of dosing suspensions were between 92 and 100% of nominal values, which is within the in-house target range of 90 to 110% of nominal concentration. The dosing suspensions were therefore considered acceptable for use on the current study.

Details on mating procedure:

- Impregnation procedure: [cohoused]
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight
- Further matings after two unsuccessful attempts: not referred to
- Verification of same strain and source of both sexes: yes, stock males of same strain from same supplier
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

GD 6 to 20

Frequency of treatment:

once daily

Duration of test:

until GD 21

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

23 pregnant females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Doses based on results obtained in a range-finding study (SA 04115), where pregnant rats received up to 400 mg/kg bw/day, from GD 6 to 20. At this dosage 1/8 females showed marked clinical signs and body weight loss. The overall mean body weight gain between GD 6-21 for this group was reduced by 29%, with mean food consumption reduced by approximately 14% between GD 16-18 and GD 18-21, compared with controls. At the limited fetal skeletal examination, a delay in ossification for several parameters was observed.
In the current study the high dose of 200 mg/kg bw/day was expected to cause slight maternal and fetal toxicity. The mid dose of 25 mg/kg bw/day provided an eight-fold factor between the high and mid dose in case 200 mg/kg bw/day should prove to be too toxic. The low dose of 10 mg/kg bw/day was expected to be a No Observed Adverse Effect Level (N.O.A.E.L.) in terms of maternal and fetal toxicity.

- Rationale for animal assignment: random

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Clinical signs daily from GD 0 through GD 21; mortality twice daily (in the morning and in the afternoon, except at weekends and public holidays when check was carried out once daily).
- Cage side observations included: all clinical signs, mortality (dead or moribund animals)

BODY WEIGHT: Yes
- Time schedule for examinations: On GD 0, 6, 8, 10, 12, 14, 16, and 18

FOOD CONSUMPTION: Yes, for periods from GD 1-6, 6-8, 8-10, 10-12, 12-14, 14-16, 16-18, and 18-21
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined:
Animals killed in extremis or found dead (1 high dose female): Macroscopic examination of visceral organs; the lungs, trachea, esophagus and samples from liver were retained in 10% neutral buffered formalin for possible histological examination. The number and type of implantations and corpora lutea were noted when present.

All surviving females were killed by carbon dioxide inhalation for examination of uterine content. Each female was first subjected to macroscopic examination of the visceral organs. The liver was weighed for all pregnant females. A portion of liver was retained in 10% neutral buffered formalin for possible histological examination, and the remaining portion stored at -80 °C for possible enzyme analysis, for all females at scheduled sacrifice. The kidneys from one animal of the low-dose group and the spleen from another one were retained in 10% neutral buffered formalin for possible histological examination.

OTHER:

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Sex of live fetuses, individual weight of live fetuses; if possible, sex and weight of dead fetuses were recorded and included in the study file, as well.
Uterine horn(s) without visible implantations were immersed in a 10% solution of ammonium sulfide according to the SALEWSKI method (1964) in order to visualize any sites which were not apparent.
Intra-uterine death was classified according to GLEICH and FROHBERG (1977) as:
- Early resorptions: macroscopic discrimination between fetal residues and placental material not possible.
- Late resorptions: distinct macroscopic discrimination between fetal and placental remains possible.

Dead fetuses: defined as dead conceptuses showing distinct digits on fore- and hind-paws.
Runt fetuses: defined as live fetuses weighing less than 4 g at cesarean section of the dam.

Fetal examinations:

- External examinations: Yes: All the live fetuses were killed by subcutaneous injection (0.02 mL/fetus) of Dolethal (18.22 g/100 mL, sodium pentobarbital) and subjected to an external examination.
- Soft tissue examinations: Yes: Approximately half of the live fetuses from each litter were stained in Bouin's fluid for subsequent internal examination following free-hand sectioning.
- Skeletal examinations: Yes: The remaining half of the live fetuses from each litter were skinned, eviscerated and stained with alizarin red S and alcian blue for skeletal examination of bone and cartilage. Structural deviations were classified as follows:
Malformations: A permanent structural change likely to adversely affect survival or health.
Variations: A change that occurs within the normal population under investigation and is unlikely to adversely affect survival or health (this might include a delay in growth or morphogenesis that has otherwise followed a normal pattern of development).
- Head examinations: No

Statistics:

Mean and standard deviation for all maternal, litter and fetal parameters were calculated for each group. Statistical analyses were performed separately for all pregnant females and for all pregnant females with live fetuses.
Only for body weight descriptive statistics were done as the only exception.

For details on statistics performed please refer to "Any other information on materials and methods incl. tables".

Indices:

Maternal body weight (BW) changes for interval periods; Corrected body weight change (CBWC); Average food consumption (FC) during intervals in g/day; Pre-implantation loss per litter; Post-implantation loss per litter; Number of live fetuses per litter; Number of dead fetuses per litter; Percentage of dead fetuses per litter; Percentage of male fetuses per litter; Mean fetal body weight per litter; Mean fetal body weight per litter and per sex; Mean fetal body weight per group; Mean fetal body weight per sex per group; Percentage of fetuses affected per litter; Mean percentage of fetuses affected per group; Percentage of litters affected per group

Historical control data:

Historical control data from studies conducted in-house were provided and referred to in order to allow comparison with concurrent controls.

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: not adverse

Details on maternal toxic effects:
No treatment-related mortality occurred. One animal of the high dose group had died due to gavage error as confirmed by necropsy. No treatment-related clinical signs were observed at any dose level. The pregnancy rate was unaffected by treatment (100% in 200 and 25 mg/kg bw/day and control group, 96% at 10 mg/kg bw/day). At 200 mg/kg bw/day, mean maternal body weight gain was reduced by 67% between GD 6-8, compared with the controls. Thereafter, mean maternal body weight gain was reduced by between 10 and 14% at each specified interval, resulting in an overall reduction of 14% between GD 6-21, compared with the controls. The effect was not statistically significant. There was no effect on mean maternal weight in any other group. At 200 mg/kg bw/day, mean maternal corrected body weight change (maternal body weight change independent of the uterine weight) was less pronounced (36.5 g) than in the control group (50.6 g), the effect being statistically significant (p≤0.05). There was no effect on mean maternal corrected body weight change at 25 or 10 mg/kg bw/day. At 200 mg/kg/day, mean maternal food consumption was reduced by between 4 and 10% at each specified interval compared with the controls, the effect being statistically significant between GD 6-8 (p≤0.01), GD 12-14 (p≤0.05) and GD 18-21 (p≤0.05). No effects were observed in the other groups. Mean maternal liver weights were unaffected by treatment. No treatment-related macroscopic findings were observed at autopsy.

Dose descriptor:

NOEL

Remarks:

maternal toxicity

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: no effects observed

Dose descriptor:

LOEL

Remarks:

maternal toxicity

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

>= 200 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: no adverse systemic effects observed

Dose descriptor:

NOEL

Remarks:

developmental toxicity

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: no effects observed on development

Dose descriptor:

LOEL

Remarks:

developmental toxicity

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

>= 200 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: no adverse effects on development observed

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes. Remark: not adverse

Details on embryotoxic / teratogenic effects:
At 200 mg/kg bw/day, mean fetal body weight for the combined sexes was reduced by 8% and by 9% and 7% in male and female fetuses, respectively, the effect being statistically significant in each case (p≤0.01). At 10 mg/kg bw/day, mean fetal body weights for the combined and separate sexes were statistically significantly lower than the control group, as well. However, in the absence of a dose-response relationsship, this finding at 10 mg/kg bw/day was considered to be incidental.
There was no effect on other litter parameters including pre and post implantation loss, early and late resorptions, fetal death status and percentage of male fetuses.
There were no treatment-related findings at the external fetal examination. The higher incidence of runt fetuses in the treated groups did not occur in a dose-related manner and was either well-comparable to or within the in-house historical control range at the high dose level; therefore, it was not considered to be treatment-related.
The same argument is valid for the observed visceral malformations like severe renal pelvic dilatation or misshapened thymus. All other variations occurred at a higher incidence in the control group compared with the treated groups, or occurred as isolated findings and were therefore considered to have occurred by chance.
The few skeletal malformations observed were distributed across the groups including the controls, with no indication of a dose-related effect, or occurred as isolated findings. These malformations were therefore considered to have arisen spontaneously.
The incidences of the observed variations (deviations in ossification), although being higher in the treated groups than in the controls at both the fetal and litter level, either lacked a dose-response relationship or were within the in-house historical control range, or both. Therefore, these findings were considered to have occurred by chance.

Dose descriptor:

NOAEL

Remarks:

embryotoxic / teratogenic effects

Effect level:

>= 200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Abnormalities:

no effects observed

Developmental effects observed:

no

CONCLUSION

There were no treatment-related mortalities or clinical signs in any dose group throughout the study. Pregnancy rate was unaffected by treatment. A dose level of 200 mg/kg/day caused slight maternal toxicity as evidenced by the reduction of mean maternal body weight gain, mean maternal corrected body weight change (maternal body weight change independent of the uterine weight), mean maternal food consumption and mean fetal body weight. Although statistically significant to some extent and very likely treatment-related, these effects were not considered adverse, as they did neither affect survival of dams or litters or their normal development nor indicate a handicap in adapting to the environment.

Therefore, a dose level of 200 mg/kg bw/day was considered as No Observed Adverse Effect Level (NOAEL) for both maternal and developmental toxicity, being the Lowest Observed Effect Level (LOEL) at the same time under the conditions of the study. A dose level of 25 mg/kg bw/day was a clear No Observed Effect Level (NOEL) in terms of both maternal and developmental toxicity.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: M.A.F.F. in Japan Notification 12 Nousan No 8147, (November, 2000)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Chatillon-sur-Chalaronne, France.
- Age at study initiation: Approx. 18 weeks.
- Weight at study initiation: 3.15-3.85 kg at mating.
- Fasting period before study: not applicable
- Housing: Individual housing of pregnant females in suspended stainless steel wire mesh cages.
- Diet: 110 C-10 pelleted animal diet from S.A.F.E. (Scientific Animal Food and Engineering, Augy, France) ad libitum. In addition, a small amount of kale was placed in the bottom of the cage of each female, once or twice per week from arrival.
- Water: Filtered and softened tap water from the municipal water supply, ad libitum.
- Acclimation period: 4 or 5 days prior to commencement of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-21
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 8/16

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous methylcellulose 400

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The appropriate amount of test substance was suspended (w/v) in an aqueous solution of methylcellulose 400 (Fluka, Mulhouse, France) at 0.5% and stored at approximately 5 °C (± 3 °C). Test formulations were prepared six times (F1 to F6) during the study.

VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.5% aqueous methylcellulose; methylcellulose is commonly used as thickener or emulsifier in various food and cosmetic applications and toxicological studies to generate appropriate consistency.
- Concentration in vehicle: 0, 2.50, 6.25, 15.00 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability of the test substance in 0.5% aqueous methylcellulose was demonstrated in study SA 04129 at concentrations of 0.127 and 250 g/L for up to 30 days (100% and 99% of nominal concentration on day 0 at 0.127 and 250 g/L, respectively, 107% and 98% of nominal concentration on day 30 at 0.127 and 250 g/L, respectively), under similar conditions to those of the current study.
Homogeneity (surface, middle and bottom) of the suspensions was checked on the first formulation (F1) for the lowest and the highest concentrations (2.5 and 15 g/L).
The mean values obtained from the homogeneity check were used as measured concentrations. In addition, the intermediate concentration of the first formulation (F1) and all concentrations of the remaining formulations (F2 to F6, taken from the surface) were checked.

Homogeneity and concentration results ranged from 92 to 102% and were, therefore, within the in-house target range (90 to 110% of nominal concentration). According to the available results, homogeneity and concentration of the test material preparations in aqueous methylcellulose were acceptable for this study.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant
- Verification of same strain and source of both sexes: yes, males of same strain from same supplier
- Proof of pregnancy: day of insemination referred to as day 0 of pregnancy

Duration of treatment / exposure:

GD 6 to 28

Frequency of treatment:

once daily

Duration of test:

until GD 29

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Dose / conc.:

60 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

23 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The range of doses was selected based on results obtained in a range-finding study (SA 04121), where pregnant rabbits were administered the test article by gavage at 0, 10, 25, 60 or 120 mg/kg bw/day, from GD 6 to 28. Due to overt maternal toxicity, the 120 mg/kg/day dose level was terminated prematurely. At the highest surviving dose level of 60 mg/kg bw/day, there was a maternal body weight loss of 0.05 kg between GD 6-8 and 0.02 kg between GD 8-10, together with a reduced maternal food consumption of between 13-35% for each interval between GD 6-18.
- Rationale for animal assignment: randomisation procedure based on body weight.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily from GD2 through GD29 for clinical signs; cages checked twice daily for dead or moribund animals or signs of abortion (once daily at weekends and public holidays)
- Cage side observations included: clinical signs, mortality, signs of abortion

BODY WEIGHT: Yes
- Time schedule for examinations: on GD 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 29

FOOD CONSUMPTION: Yes, on day 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 29 and for periods from GD 3-4, 4-5, 5-6, 6-8, 8-10, 10-12, 12-14, 14-16, 16-18, 18-20, 20-22, 22-24, 24-26, 26-28, 28-29.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined:
Animals euthanised by i.v. injection of Dolethal (18.22 g/100 mL, sodium pentobarbital) due to moribund condition including those having aborted and animals found dead were subjected to macroscopic examination of the visceral organs; furthermore the number of ribs was recorded. The liver of all these animals were fixed in 10% neutral buffered formalin and retained for possible histological examination. The number and type of implantations and corpora lutea were noted when present. In case of no visible uterine implants, uterine horn(s) were immersed in a 2% solution of sodium hydroxide for approximately 2 hours followed by immersion in 10% buffered formalin for a further 2 hours (Sumida, 1994), in order to visualize any sites which were not apparent.

On GD 29, all surviving females were killed by intravenous injection of Dolethal (18.22 g/100 mL, sodium pentobarbital) for examination of uterine content. Each female was first subjected to macroscopic examination of the visceral organs, and the number of ribs was recorded. The liver of all pregnant females was weighed, this tissue was retained in 10% neutral buffered formalin from all females for possible histological examination.

Ovaries and uterine content:

The ovaries and uterine content were examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Number of live and dead fetuses, individual weight of fetuses, body weight of dead fetuses if possible. Uterine horn(s) without visible implantations were immersed in a 2% solution of sodium hydroxide for approximately 2 hours followed by immersion in 10% buffered formalin for a further 2 hours (Sumida, 1994) in order to visualize any sites which were not apparent.

Intra-uterine resorption death was classified according to GLEICH and FROHBERG (1977) as:
- Early resorptions: macroscopic discrimination between fetal residues and placental material not possible.
- Late resorptions: distinct macroscopic discrimination between fetal and placental remains possible.

Dead fetuses: defined as dead conceptuses showing distinct digits on fore and hind-paws.
Runt fetuses: defined as live fetuses weighing less than 28 g at cesarean section of the dam.

Fetal examinations:

- External examinations: Yes: All live fetuses were killed by subcutaneous injection (0.1 mL/fetus) of Dolethal (18.22 g/100 mL, sodium pentobarbital) and subjected to an external examination.
- Soft tissue examinations: Yes: The bodies of all fetuses were dissected for soft tissue anormalities and sexed.
- Skeletal examinations: Yes: After soft tissue examination all fetuses were fixed in absolute ethanol before staining (a modification of the Staples and Schnell staining technique was performed).
- Head examinations: Yes: After internal examination of the neck, the head of fetuses from approximately half of each litter was immersed in Bouin's fluid and the internal structures examined after fixation.

Statistics:

Mean and standard deviation for all maternal, litter and fetal parameters were calculated for each group. Statistical analyses were performed separately for all pregnant females and for all pregnant females with live fetuses.
Only for body weight descriptive statistics were done as the only exception.

For details on statistics performed please refer to "Any other information on materials and methods incl. tables".

Indices:

Maternal body weight (BW) changes for interval periods; Corrected body weight change (CBWC); Average food consumption (FC) during intervals; Pre-implantation loss per litter; Post-implantation loss per litter; Number of live fetuses; Number of dead fetuses; Percentage of dead fetuses per litter; Percentage of male fetuses per litter; Mean fetal body weight per litter; Mean fetal body weight per litter and per sex; Mean fetal body weight per group; Mean fetal body weight per sex per group; Percentage of fetuses affected per litter; Mean percentage of fetuses affected per group; Percentage of litters affected per group

Historical control data:

Historical control data from studies conducted in-house were provided and referred to in order to allow comparison with concurrent controls.

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: treatment-related

Mortality:
At 60 mg/kg bw/day, one female was killed for animal welfare reasons on GD 27 after having aborted. Clinical signs consisted of few feces on GD 26 and 27, a 0.17 kg loss in body weight together with a reduced food intake between GD 24-26. At autopsy this female was noted to have a pale liver. The relevance to treatment of these findings is unclear.

At 25 mg/kg bw/day, there were no deaths or premature sacrifices.

At 10 mg/kg bw/day, one female was killed for animal welfare reasons on GD 22. This female had few/no feces between GD 16-22, had lost 0.37 kg in body weight and had eaten very little between GD 12-22. No treatment-related findings were noted at autopsy for this female. In the absence of maternal toxicity at the higher dose level of 25 mg/kg bw/day, these findings in this single female were considered not to be related to treatment. Another female was sacrificed on GD 12 due to a gavage error. There were no in-life findings for this female, at autopsy liquid was found in the trachea. One female of the control group was sacrificed on GD 21 due to assumed gavage error. There were no in-life findings for this female, at autopsy the lungs were found to be mottled with multiple foci.

Clinical observations:
At 60 mg/kg bw/day, 3/23 females had mucoid feces on two or more occasions from GD 16 onwards, compared with no cases in the control group. In addition, 12/23 females had few feces on one or more occasions, compared with 2/23 control females. At 25 and 10 mg/kg bw/day, few feces were observed in 5/23 females and 9/23 females, respectively. The incidence of this finding was very similar at 25 mg/kg bw/day and in the control group. This observation did not occur in a dose-related manner at 10 and 25 mg/kg bw/day, and is a very common spontaneous finding in the strain of rabbit used under similar conditions; therefore, this finding was considered to be incidental at the two lower dose levels.
All other clinical signs occurred as isolated findings distributed across the groups, and were therefore considered to be incidental.

Body weights:
At 60 mg/kg bw/day, there was a 0.03 kg loss in mean maternal body weight between GD 6-8, compared with a static body weight in the control group, the effect was not statistically significant. Thereafter, a mean maternal body weight gain was reduced at most intervals, resulting in an overall reduction of 30% (p≤0.05) between GD 6 -29, when compared with controls. Mean maternal corrected body weight change (body weight - gravid uterine weight) was -0.24 kg (p≤0.01) compared with -0.08 kg in the control group.
At 25 and 10 mg/kg bw/day, mean maternal body weight and mean maternal corrected body weight change were unaffected by treatment.

Food consumption:
At 60 mg/kg bw/day, mean maternal food consumption was reduced by between 10 and 30% at each interval compared with the controls, the effect being statistically significant (p≤0.05 or p≤0.01) at most intervals. At 25 and 10 mg/kg bw/day, mean maternal food consumption was similar to the controls.

Gross pathology and organ weights:
There was no effect on mean maternal liver weight at any dose level tested. At 60 mg/kg bw/day, treatment-related macroscopic findings consisted of pale liver in 3/23 females and white foci on the liver in 2/23 females, compared with no cases in the controls. There were no treatment-related macroscopic findings at 25 or 10 mg/kg bw/day.

Cesarian section:
Pregnancy rate was unaffected by treatment, with a pregnancy rate of 96% at 60 mg/kg bw/day, 100% at 25 mg/kg bw/day, 83% at 10 mg/kg bw/day and 87% in the control group.
There was no treatment-related effect on any litter parameter assessed, including mean fetal body weight, number of live fetuses, early or late resorptions, percentage of male fetuses and percentage of pre or post-implantation loss.
At 10 mg/kg bw/day, the percentage of dead fetuses per litter (9.0%) was higher than in the control group (2.9%) and outside the in-house historical control range (HCD) of 0.74 to 6.58%. However, as the percentage of dead fetuses per litter at the two higher dose levels of 25 and 60 mg/kg bw/day, was 4.4% and 5.5%, respectively, which is considerably lower than at 10 mg/kg bw/day, only marginally higher than in the controls and well within the HCD, the finding at 10 mg/kg bw/day, was considered to have occurred by chance.

Dose descriptor:

NOEL

Remarks:

maternal toxicity

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: no effects observed

Dose descriptor:

LOAEL

Remarks:

maternal toxicity

Effect level:

60 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

gross pathology

number of abortions

Dose descriptor:

NOEL

Remarks:

developmental toxicity

Effect level:

>= 60 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: no effects on development observed

Abnormalities:

effects observed, treatment-related

Description (incidence and severity):

One female aborting, the animal was sacrificed thereafter on GD 27.
Furthermore, a loss in body weight or reduced body weight gain as well as reduced food consumption and macroscopic changes in the liver (3/23 pale, 2/23 white foci) were observed at 60 mg/kg bw/day.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There was no treatment-related effect on any litter parameter assessed, including mean fetal body weight, number of live fetuses, early or late resorptions, percentage of male fetuses and percentage of pre or post-implantation loss. There were no treatment-related malformations or variations noted at the external, visceral and the skeletal fetal examination.

Fetal evaluation:
External observations:
There were no treatment-related malformations or variations noted at the external fetal examination. The four malformations observed (malrotated hindpaw; skull, neck and thoracic wall: edema; spina bifida (aperta); omphalocele) occurred as isolated findings distributed between the mid- and low-dose group. The incidence of runt fetuses was similar between the treated and control groups. The two other variations observed (short thoracic cavity, hematoma of the skull) occurred as isolated findings at the low-dose only.

Visceral observations:
There were no treatment-related malformations or variations noted at the visceral fetal examination.
The few malformations observed occurred as isolated findings distributed between mid- and low-dose and control group, together with two cases of the malformation "ocular coloboma" in the control group and one case in the mid-dose group. At 60 mg/kg bw/day, the incidence of the variation "thymic remnant present (unilateral/bilateral)" was slightly outside the HCD at the litter and fetal level, but as the incidence was very similar in the concurrent control group, this common finding was considered to have occurred by chance. The remaining variations noted occurred at a similar incidence between the treated and control groups, at a very low frequency or in a non dose-related manner.

Skeletal observations:
There were no treatment-related malformations or variations at the skeletal fetal examination. The malformations observed occurred as isolated findings distributed across the treated and control groups. The incidences of variations comprising incomplete ossification, 13th thoracic rib detached and presence of 27 presacral vertebrae were either not dose-related or inside the ranges of the historical control data. Incidences which were higher than in the control group and marginally outside the historical control data (13th thoracic rib unilateral/bilateral) were statistically analysed. In the absence of a statistically significant effect, this finding was considered to have occurred by chance.

Dose descriptor:

NOEL

Remarks:

embryotoxic / teratogenic

Effect level:

>= 60 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Abnormalities:

no effects observed

Developmental effects observed:

no

CONCLUSION:

A dose level of 60 mg/kg bw/day of the test material administered to the pregnant New Zealand White rabbit by oral gavage resulted in maternal toxicity as evidenced by one female aborting, a loss in or reduced body weight gain and food consumption, and macroscopic changes in the liver. Fetal development was unaffected by treatment at any dose level tested. A dose level of 25 mg/kg bw/day was considered to be a No Observed Effect Level (NOEL) in terms of maternal toxicity, whilst a dose level of 60 mg/kg bw/day was considered to be a NOEL in terms of developmental toxicity. Considering the observations at 60 mg/kg bw/day, this dose has to be considered as Lowest Observed Adverse Effect Level (LOAEL) for maternal toxicity under the conditions of this study.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 1) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.7, of Regulation (EC) No 1907/2006.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Two studies are available addressing prenatal developmental toxicity of the test substance. The studies were conducted in rats and rabbits, respectively, according to OECD guideline 414 under GLP conditions.

In the first study rats were dosed with test substance doses of 10, 25 and 200 mg/kg bw/day from gestation day 6 until gestation day 20 by oral gavage. No treatment-related mortality occurred, and no treatment-related clinical signs were observed at any dose level tested in the dams. The only effects observed were slight reductions in maternal body weight gain and statistically significant reductions in food consumption at specific treatment intervals. No effects were observed in the other groups, and there were no treatment-related macroscopic findings at autopsy.

In the offspring there were slight but statistically significant reductions in mean fetal body weight in the high-dose and in the low-dose group at Cesarian section. However, in the absence of a dose-response relationship the findings in the low dose group were considered to be incidental. There were no other treatment-related effects on litter parameters observed, and there were no treatment-related findings at the external examinations. The few observations made, like runt fetuses, visceral or skeletal malformations and deviations in ossification, did either not occur in a dose-related manner, were well-comparable with the provided historical controls, or occurred as isolated findings well-distributed across the groups including the controls. Therefore, a dose level of 200 mg/kg bw/day was considered as NOAEL for both maternal and developmental toxicity in rats. This dose level at the same time represented the LOEL under the conditions of the study. A dose level of 25 mg/kg bw/day was detemined as NOEL for both maternal and developmental toxicity.

In the second study test substance doses of 10, 25 and 60 mg/kg bw/day were administered to rabbits from gestation day 6 until gestation day 28 by oral gavage. One female in the high-dose group aborted and was sacrificed thereafter on GD 27. Furthermore, effects on body weight and body weight gain as well as food consumption were observed. At autopsy, macroscopic changes (pale liver, white liver foci) were found in a few animals at this dose level. No treatment-related changes were observed in the mid- and low-dose groups. At Cesarian section there was no treatment-related effect on any litter parameter assessed. There were no treatment-related malformations or variations noted at the external, visceral and skeletal examination of the fetuses. Based on these findings a dose level of 60 mg/kg bw/day was determined to be the LOAEL for maternal toxicity in rabbits under the conditions of the study. The dose level of 25 mg/kg bw/day was considered to be the NO(A)EL for maternal toxicity. No effects were observed in the offspring, therefore the NO(A)EL for developmental toxicity was considered to be greater than 60 mg/kg bw/day in rabbits under the conditions of the study.

Justification for classification or non-classification

Adverse effects on fertility were only observed in conjunction with severe effects of systemic toxicity and were therefore considered to be secondary non-specific consequences of the other toxic effects. No developmental toxicity was observed.

According to Regulation (EC) No 1272/2008 the test substance does not have to be classified as human reproductive toxicant.